A number of optical methods have demonstrated microscopic resolutions beyond the diffraction limit. These methods, which all requiring fluorescent labeling, include Scanning Near-field Optical Microscopy (SNOM) (see I. Horsh et al. “A stand-alone scanning near-field optical microscope,” Photons and Local Probes, NATO ASI Series E:300:139 (1995)), Stimulated Emission Depletion (STED) (see S. Hell et al. “Breaking the diffraction resolution limit by stimulated-emission—stimulated-emission-depletion fluorescence microscopy,” Optics Letters. 19:495 (1995)) and Ground State Depletion (GSD) (see S. Hell et al. “Ground-State-Depletion fluorescence microscopy—a concept for breaking the diffraction resolution limit,” Applied Physics B. 60:780 (1994)) fluorescence microscopy, photo-activated localization microscopy (PALM) (see E. Betzig et al. “Imaging intracellular fluorescent proteins at nanometer resolution,” Science 313:1642 (2006)), stochastic optical reconstruction microscopy (STORM) (see M. Rust et al. “Sub-diffraction-limited imaging by stochastic optical reconstruction microscopy (STORM),” Nature Methods 3:783 (2006)), and structured illumination microscopy (SIM) (see B. Bailey et al. “Enhancement of Axial Resolution in Fluorescence Microscopy by Standing-Wave Excitation,” Nature 366:44 (1993); see M. Gustafsson “Surpassing the lateral resolution limit by a factor of two using structured illumination microscopy,” Journal of Microscopy 198:82 (2000); and see M. Gustafsson “Nonlinear structured illumination microscopy: Wide-field fluorescence imaging with theoretically unlimited resolution,” PNAS 102:13081 (2005)). In addition, it is possible to improve the position accuracy beyond the diffraction limit in linear fluorescence microscopy and two-photon microscopy (see W. Denk et al. “2-Photon Laser Scanning Fluorscence Microscopy,” Science 248:73 (1990)) by fitting the point spread function (see R. Thompson et al. “Precise nanometer localization analysis for individual fluorescent probes,” Biophysical Journal 82:2775 (2002)).
Optical coherence tomography, Spectral Domain OCT and Optical Frequency Domain Imaging include certain imaging techniques that measure the interference between a reference beam of light and a detected beam reflected back from a sample. A detailed system description of traditional time-domain OCT has been described in D. Huang et al., “Optical Coherence Tomography,” Science 254: 1178 (1991). Exemplary detailed descriptions for spectral-domain OCT and optical frequency domain interferometry systems are provided in U.S. patent application Ser. Nos. 10/501,276 and 10/577,562, respectively.
Another exemplary technique that can achieve resolution beyond the diffraction limit can be called self-interference fluorescence microscopy (SIFM). This exemplary technique is related to the work described in K. Drabe et al. “Localization of Spontaneous Emission in front of a mirror,” Optics Communications 73:91 (1989) and Swan et al. “Toward nanometer-scale resolution in fluorescence microscopy using spectral self-interference,” IEEE Quantum Electronics 9:294 (2003). It has been demonstrated that the position of a fluorophore located in front of a reflecting surface can be determined with nanometer resolution by analyzing the self-interference of the emitted fluorescence light with the reflection from the surface. The exemplary technique is related to Optical Coherence Phase Microscopy (OCPM), another technique derived from OCT (see C. Joo, et al. “Spectral-domain optical coherence phase microscopy for quantitative phase-contrast imaging,” Optics Letters 30:2131 (2005); and see C. Joo, et al. “Spectral Domain optical coherence phase and multiphoton microscopy,” Optics Letters 32:623 (2007)), where the phase of the interference between a sample and a reference arm is used to determine motion on a (sub) nanometer length scale.
In OCPM, an external light source is used, and the light is scattered by structures in tissue or within a cell. OCPM has demonstrated a phase sensitivity corresponding to 25 picometers. In SIFM, the light source is the fluorophore itself, which is excited by an excitation source. Fluorescent light emitted in different directions is captured and made to interfere with itself after passing through a spatial phase retarding optical element. The interference can be spectrally resolved in a spectrometer, generating an interference pattern with a periodicity corresponding to the path length differences experienced by the light traversing the phase retarding element. A Fourier transform of the spectrally resolved interference may generate a profile as in Spectral Domain Optical Coherence Tomography (SD-OCT), with a point spread function determined by the fluorescence bandwidth. By using the phase term of the transform, the fluorophore can be localized with a resolution far better than the diffraction limit. This exemplary approach generally makes use of a single microscope objective and a specially designed wave plate that collect the emitted light the epi-direction with a high numerical aperture and dividing the beam in 4 sections, each with different delays, for three-dimensional triangulation.
There may be a need to overcome certain deficiencies associated with the conventional arrangements and methods described above.